The importance of brain-derived G protein-coupled receptors (GPCRs) in mental function and health can hardly be overstated. However, our knowledge of the molecular biophysical basis for how GPCRs bind to their cognate agonists and antagonists to elicit function is limited by the lack of high resolution 3-D structural information for these proteins. In this proposal we seek to directly address a formidable general barrier to structural determination: the need for membrane mimetic media that are suitable both to confer aqueous solubility to GPCRs (as required by solution NMR and X-ray crystallographic methods) and to sustain native-like receptor structure, function, and stability. In preliminary data for the human kappa opioid receptor (KOR), we show that the receptor can be highly overexpressed and purified to homogeneity. However, the preliminary structural data fails to establish the feasibility of conduction high resolution structural studies. We hypothesize that the challenges confronting structural analysis can be overcome through the development and application of two novel classes of amphiphiles: two-headed "birosh" detergents and micelle-soluble cholesterol derivatives. The test system for these studies is the purified KOR. However, the methods and detergents developed herein should be generally applicable to other brain-derived GPCRs. The results of this work will positively impact not only structural analysis of GPCRs, but will also be useful in other applications that benefit from abundant quantities of pure and properly-folded GPCRs, including drug screening, biochemical studies, and studies of receptor stability and folding. [unreadable] Specific Aim 1. Develop a novel class of detergents ("birosh" detergents) that will self-assemble with more traditional detergents in aqueous solution to form a new class of bicelles that confer native stability and structure to solubilized KOR and other GPCRs. These two-headed detergents are designed to be much better than conventional detergents at promoting the native-like stability and functionality of solubilized GPCRs, providing the basis for breakthroughs in receptor structural biology. [unreadable] Specific Aim 2. Develop novel micelle-soluble cholesterol derivatives can be used to enhance the solubility and functionality of the micelle-solubilized KOR and other GPCRs. A number of receptors and other membrane protein appear to require interactions with cholesterol in the membrane in order to be fully functional. However, cholesterol is notoriously difficult to solubilize in the micellar conditions typically used for membrane proteins. To allay this limitation, we propose to synthesize and characterize several novel cholesterol derivatives. [unreadable] [unreadable] [unreadable]